Comprehensive analysis of key cancer proteins and pathway markers in clinical samples remains challenging yet is critical in assessing efficacy of molecularly targeted drugs in clinical trial and in understanding complex tumor biology. Currently, the number of markers being studies is often limited (<10) and requires time- consuming analyses of tissue sections harvested by large core biopsies which carry a not insignificant morbidity. We developed a technology that allows simultaneous analysis of hundreds of proteins in cancer cells harvested from fine needle aspirates (FNA). The method capitalizes on DNA-barcoded antibody sensing where barcodes are photo-cleaved and digitally detected without any amplification steps. In a recent proof-of- concept study (Sci Transl Med 2014;6: 219ra9) this method showed high reproducibility, achieved single cell sensitivity and was able to identify pathway responses to molecularly targeted drugs, even in single cells. Compared to existing technology (immunohistochemistry, cytometry and mass spectrometry) it: i) allows hundreds of markers to be detected simultaneously, ii) works well in single cells or small numbers of cells, iii) does not destroy valuable samples, iv) is fast and inexpensive and v) can be combined with mRNA and DNA analytical techniques. The goal of this R33 application is to further the technology by integrating it with i) on-chip microfluidics for cancer cell enrichmen and single/bulk cell harvesting and ii) combined protein, mRNA and DNA analysis. We will then expand and rigorously test this next generation device for point-of-care analyses of single cells in two clinical studies to broadly demonstrate its broad cancer utility: i) in a clinical diagnosti study to compare cancer cell protein profiles in breast cancer patients and ii) in a drug trial of PI3K inhibition to determine treatment response/failure over time. The proposed integrated profiling method has the potential to transform cancer research and clinical practice. It will allo inexpensive, more extensive and robust profiling of cellular markers in scant and valuable materials from clinical trials.